The traditional method for determining the progression of periodontitis is to assess the degree of damage which has occurred to the periodontal tissues over a given period of time by probing. Unfortunately, as a measurement technique, periodontal probing has several sources of error which make it inexact.
Radiographs have long been used to assess the level and loss of alveolar bone around teeth. However, it is impossible to visually detect minor changes in bone. Hence, the amount of bone destruction/loss tends to be underestimated when radiographs are read in a routine fashion.
In the past decade there has been increased interest in developing, testing and refining diagnostic aids for the early detection of the presence and progression of periodontitis. It is evident that any diagnostic and/or prognostic test for progressing periodontal lesions should provide information that offers some advantage over the conventional indicators of disease.
Data from epidermiological studies have confirmed that periodontal diseases are multifactorial in nature (Armitage, G. C., CDA Journal 36: 35-41, 1993). This data indicates that periodontal diseases are caused by an infection with one or more of a handful of periodontopathogenic microbes. These organisms include Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Bacteroides forsythus (Bf), Actinobacillus actinomycetemcomitans (Aa), Campylobacter rectus, Fusobacterium nucleatum and spirochetes.
Much work, without giving definitive and clear resolution, has been done to determine the specific groups of subgingival microorganisms which are most strongly associated with progressing lesions (Armitage, G. C., CDA Journal 36: 35-41, 1993). The target microorganisms have been assessed by enzyme tests, culture analysis, microscopic analysis and DNA-probes.
Gingival crevicular fluid (GCF) is an inflammatory exudate that flows into the oral cavity from periodontal pockets. It contains subgingival bacteria, inflammatory cells and a lot of different substances produced by bacteria as well as host cells present. It is an inflammatory exudate with several inflammatory reactions occuring in the adjacent periodontal tissues (Birkedal-Hansen, H., J. Periodontol. 64: 474-484, 1993). In clinical practice, GCF is easy to collect by placing filter paper strips at the pocket orifice. These features have made GCF an attractive source of potential markers for the progression of periodontitis. These markers include: 1) products associated with tissue remodeling and breakdown, 2) inflammatory mediators and 3) host cell-derived enzymes (Birkedal-Hansen, H., J. Periodontol. 64: 474-484. 1993).
One of the major features of periodontitis is the destruction of connective tissue and bone. Abundant amounts of tissue breakdown products from these tissues are released during the periodontal disease process. In cross-sectional studies, GCF collected from sites with periodontitis have been shown to contain elevated levels of hydroxyproline from e.g. collagen degradation (Talonpoika, I., Changes in the composition of gingival crevicular fluid after periodontal treatment, Thesis, Ann. Univ. Turku 142, 1994). However, longitudinal studies have not been done to determine if the presence of these substances is strongly associated with progressive lesions. It is not yet known if these products are associated with the actual periodontal tissue destruction or remodeling.
Many different inflammatory mediators are produced by periodontal tissues affected by gingivitis and periodontitis. Some of these mediators appear to play a central role in the destructive processes observed in cases of periodontitis, which has led some investigators to examine the possibility of using certain inflammatory mediators as markers for progressive lesions (Sorsa, T. et al. Arch. Oral. Biol. 35: 193S-196S, 1990), Page, R. C., J. Periodont. Res. 26: 230-242, 1991).
However, so far there are no fully developed and validated diagnostic or prognostic tests for progressive periodontitis which would assess the level of inflammatory mediators in GCF. Preliminary work has suggested that the following substances may be associated with sites afflicted with periodontitis: prostaglandin E.sub.2 and tumor necrosis factor-.alpha. (Page, R. C., J. Periodont. Res. 26: 230-242, 1991). However, all these inflammatory mediators are also strongly associated with bone resorption in other parts of the body. It is likely that they are all involved in the progression of periodontitis, but additional studies are needed to determine whether they can serve as useful markers for disease progression in a clinical setting.
A great deal of work has been done on developing tests for enzymes in GCF that may be related to the progression of periodontitis. Among the host-derived enzymes present in GCF that have received the most attention are: aspartate aminotransferase and collagenase (Page, R. C., J. Periodont. Res. 26: 230-242, 1991) gelatinase (Birkedal-Hansen, H., Crit. Rev. Oral. Biol. Med. 4: 197-250, 1993 and Sorsa, T., et al., Ann. N.Y. Acad. Sci. 732: 112-131, 1994) and related neutral proteases (Teng, Y. T., et al., J. Periodont. Res. 27: 544-552, 1993) .beta.-glucoronidase (Page, R. C., J. Periodont. Res. 26: 230-242, 1991) and elastase (Ingman, T., et al, Oral. Microbiol. Immunol. 8: 298-305, 1993). Some of these enzymes are released from dead and dying cells of the periodontium, some come from polymorphonuclear neutrophils (PMN), and others are produced by other inflammatory cells (PMNs/monocytes/macrophages), epithelial and connective tissue cells at affected sites (Sorsa, T., et al., Arch. Oral Biol. 35: 193S-196S, 1990, Birkedal-Hansen, H., et al., Crit. Rev. Oral. biol. Med. 4: 197-250, 1993, Sorsa, T., et al. Ann. N.Y. Acad. Sci. 732: 112-131, 1994).
Aspartate aminotransferase (AST), formerly called glutamic oxalotransferase (GOT), is released by dead and dying host cells. In medicine, it is a useful marker for the cell death that occurs in cardiac muscle after a myocardial infarction or in the liver during hepatic disease. In fact, it is released from the dead cells of virtually all tissues of the body. Results from several longitudinal studies of patients with progressive periodontitis, in which increased clinical attachment loss was used as the criterion for progression, suggest that GCF AST levels might serve as a marker for disease progression on a site-by-site basis (Page, R. C., J. Periodont. Res. 26: 230-242, 1991). A rapid chair-side test for the enzyme in GCF has been developed. AST, based on enzymatic activity determinations, is also somewhat elevated at sites with gingivitis and nonprogressing periodontitis. Therefore, it remains to be established whether its GCF levels can usefully distinguish between inflamed sites that are breaking down and those which are not (Page, R. C., J. Periodont. Res. 26: 230-242, 1991). Thus, the use of an AST assay does not give an unambiguous diagnosis of periodontitis. Additionally, the enzymatic assay is inexact due to possible AST derived from blood contaminating the GCF.
Polymorphonuclear neutrophilic leukocytes (PMNs) are prominent inflammatory cells found in GCF from sites with gingivitis or periodontitis. They are the first line of defense against bacteria that colonize periodontal pockets. PMNs release a wide range of lysosomal and subgranular enzymes, inflammatory mediators and proteinases when challenged by bacteria. The logic behind the idea that some of these enzymes might serve as markers for the progression of periodontitis is simple. That is, when periodontitis progresses, subgingival bacteria overwhelm local host defenses (including PMNs) and lysosomal enzymes, proteases/inflammatory mediators from dead neutrophils are released into the GCF in abundance. A rapid chair-side test for a group of the lysosomal enzymes (i.e. neutral proteases) has been developed (Periocheck.TM.)). They have been shown to be elevated in GCF from sites with advanced periodontitis (Teng, Y. T., et al., J. Period. Res. 27: 544-552, 1993). However, Periocheck.TM.) is not sufficiently specific for PMN proteases and microbial proteinases can well degrade the substrate included into it. Neither has it been longitudinally tested to determine if it can identify sites that are at an increased risk for progression.
Preliminary work from one longitudinal study has shown that GCF levels of another lysosomal enzyme, .beta.-glucuronidase, were elevated in patients in whom increased clinical attachment loss was detected over a one-year observation period (Armitage, G. C., CDA Journal 36: 35-41, 1993, Page, R. C., J. Periodont. Res. 26: 230-242, 1991). Lower levels of the enzyme were found in patients with nonprogressing disease. However, no rapid chair-side test for .beta.-glucuronidase in GCF has been developed and further it remains to be determined, whether a rapid chair-side GCF test for .beta.-glucuronidase can be developed into a useful method for detecting, on a site-by-site basis, the progression of periodontitis.
Elastase is one of the prominent neutral serine proteinases released into GCF by PMNs. However, it is known that other cells produce said enzyme (McCullough, C. A. G., J. Clin. Periodontol. 21:. 497-506, 1994). A rapid chair-side test for the enzyme in GCF has been developed based on enzymatic activity and partially tested in a clinical setting (McCullough, C. A. G., J. Clin. Periodontol. 21: 497-506, 1994). Longitudinal studies of patients with untreated periodontitis, in whom additional bone loss as detected by subtraction radiography was used as the criterion for progression, have suggested that elevated elastase levels in GCF are strongly associated on a site-by-site basis with the progression of periodontitis (McCullough, C. A. G., J. Clin. Periodontol. 21: 497-506, 1994). It was found that sites with high GCF elastase levels were more likely to develop additional bone loss within six months than sites with low levels of the enzyme (McCullough, C. A. G., J. Clin. Periodontol. 21: 497-506, 1994).
Although enzymatic GCF-based tests appear to be promising, much more work needs to be done before they have been fully and specifically tested and validated.
The tests developed so far and based on PMN-proteins especially elastase lack specificity; elastase is assayed by a synthetic protease substrate (SAAVNA-peptide) that lacks specificity, since it can be efficiently degraded by both human and bacterial peptidases/proteases/proteinases. Thus, a positive result in the enzymatic test may be obtained (Sorsa, T., et al., J. Periodont, Res. 22: 375-380, 1987; Ingman, T. et al., Oral. Microbiol. Immunol. 8: 298-305, 1993) even if no elastase is present in the tested sample. Such a test cannot be regarded as being conclusive for a diagnosis of the progression of periodontic disease.
In the Finnish patent application FI 943939 novel methods and test kits for a rapid and reliable, chair-side diagnosis of active periodontal disease related to bone destruction/and loss based on the determination of active human matrix metalloproteinase-8 (MMP-8)-neutrophil collagenase are described. In said patent application FI 943939, the entire disclosure of which is enclosed herein by reference, also other more conventional methods for diagnosing periodontal disease activity are extensively discussed.
Naturally, it is very important to be able to make a reliable diagnosis of the active disease with a chair-side test. For the prevention of periodontitis, it is, however, very important to be able to make a reliable prognosis for the progress of the disease, preferably by a chair-side test performed by the dentist in his office.
Kjeldsen L., et al. (J. Biol. Chem. 268: 10425-10432, 1993) have recently described the purification, characterization and primary structure of a novel neutrophil gelatinase-associated lipocalin (NGAL) from human peripheral blood PMNs. NGAL is a 25 kD protein associated with human PMN 92 kD gelatinase, i.e. matrix metalloproteinase-9 (MMP-9). Activated PMNs degranulate the 25 kD NGAL mostly from the secondary PMNs (specific) granules (Kjeldsen, L. , et al., J. Biol. Chem. 268: 10425-10432, 1993). Outside the PMNs NGAL can form covalent 120 kD complexes with 92 kDa MMP-9 degranulated from the PMN tertiary or C-type granules ((Kjeldsen, L., et al., J. Biol. Chem. 268: 10425-10432, 1993). This NGAL/MMP-9-complex formation explains the 120 kD form of MMP-9, which is unique for PMNs and their gelatinase (MMP-9) (Sorsa, T., et al., Arch. Oral Biol. 35: 193S-197S, 1990, Kjeldsen L., et al. (J. Biol. Chem. 268: 10425-10432, 1993). In addition, NGAL exists in a monomeric 25 kD and a dimeric 46 kD forms, which both can be exocytosed upon PMN activation (Kjeldsen, L., et al., J. Biol. Chem. 268: 10425-10432, 1993). NGAL has no modulatory actions on gelatinase, and the function of NGAL has not yet been completely clarified.
PMNs are the major sources of metallomatrix proteins (MMPs) (Sorsa, T. et al. J. Periodont. Res. 23: 380-393, 1988, sorsa, T., et al. Ann. N.Y. Acad. Sci. 732: 112-131, 1994, Golub, L. M., et al., J. Clin. Perio. 22: 100-109, 1995) and factors related to them, such as NGAL and consequently of the specific 120 kD complex NGAL/MMP-9 especially in periodontitis. Though GCF from periodontitis sites of HIV(+)-patients have been shown to contain increased amounts of active PMN derived MMPs (Salo, et al., Ann. N.Y. Acad. Sci. 732: 476-478, 1994), it has not previously been shown that these PMN derived proteins include NGAL, too. No reliable chair-side tests have been developed for indicating the progression of periodontitis based on PMN derived proteins.
Although, a vast amount of work has been done to provide a satisfactory method for diagnosing the progression of periodontitis, none of the developed and suggested solutions seem to be conclusive enough. To the present day, therefore, the clinician is still forced to rely heavily on the old conventional methods of assessing periodontitis and gingivitis by local probe measurements and radiographs. Moreover, none of the tests developed so far are capable of indicating a risk of periodontal disease at any degree. They are rather based on assessing elevated enzymatic activities in the mouth, which elevated activities may well be caused by quite different sources, such as tonsillitis, Sjogren syndrome, etc. In other words the enzymatic tests described above do not unambigously show that the actual and only cause of the elevated amounts of the enzyme activities is periodontal diseases. The elevated amounts might be caused by bacteria as well.
A mature disease is, as already pointed out above, often discernible by the clinician with the naked eye. The need in the art is to obtain a rapid sensitive and exact way of assessing an increased risk for a progressing periodontal disease before any visual signals are available.
There is also a clear need among dentists to obtain a sensitive, specific and rapid test for chair-side assessing how far a diagnosed periodontal disease has progressed and, later on, to monitor and assess the effects of periodontal treatment.
The present inventors have now realized that the long felt need in the art can be satisfied by providing an assay which is capable of giving a warning of an increased risk of periodontal disease and indicating the level of progression of such a disease at an early stage by monitoring the existence of activated PMNs by specific immunochemical means. Some of the previously known PMNs marker proteins such as MMP-9, elastase and myeloperoxidase are not sufficiently specific, because they can be produced by bacteria (elastase) and other cells (monocytes/macrophages) and epithelial cells produce myeloperoxidase and MMP-9 as well. According to the invention there is provided a rapid immunological test for a chair-side detection and determination of a new specific PMN marker protein, i.e. NGAL from GCF of a patient for making the diagnosis and a proper prognosis of the risks for developing a periodontal disease. Said NGAL is assessed with the aid of an immunological chair-side kit from a GCF sample taken from the patient or a saliva or mouth-rinse sample from said patient. According to the invention said NGAL serves as a biochemical adjunctive marker to monitor the course and treatment of different forms of periodontal diseases including HIV(+)-infection associated periodontitis and peri-implantitis as well as to control periodontal and peri-implant health. NGAL is especially useful because it indicates the risk at an early stage of inflammation and not only the active disease and its specificity is not disturbed by the fact that NGAL sometimes forms complexes with MMP-9.
Thus, the objective of the present invention is to provide methods for making a rapid, specific and reliable diagnosis and predicting and assessing different forms of periodontal diseases and especially the risks for said diseases by a rapid, reliable chair-side test based on the determination of a specific PMN marker, NGAL, by using specific immunochemical methods. The present invention also provides test kits, which can be used in said methods to make a diagnosis and prognosis at an early stage of the disease.